We are using protein mass spectrometry, DNA microarrays, mathematical modeling and physiological methods to investigate the mechanisms involved in regulation of water transport across the renal collecting duct epithelium. The peptide hormone vasopressin regulates water transport largely through regulation of the molecular water channel aquaporin-2. Vasopressin regulates aquaporin-2 in a time frame of seconds to minutes by altering the distribution of aquaporin-2 between the plasma membrane and the cytoplasm via vesicular trafficking. (Trafficking to the plasma membrane renders the cells permeable to water.) Current studies are aimed at discovering the endosomal compartments that contain aquaporin-2 and the proteins in these compartments that are regulated by vasopressin. We are also carrying out global profiling of signaling mechanisms in the renal collecting duct using quantitative protein mass spectrometry-based proteomics for large-scale assessment of changes in post-translational modifications (esp. phosphorylation), intracellular localization, abundance, and binding to other proteins. Vasopressin also regulates aquaporin-2 abundance in the collecting duct with a response time of hours to days. DNA array techniques are being used to investigate the roles of transcriptional regulation, regulation of protein stability and regulation of translation in long-term regulation of aquaporin-2 abundance. Additionally, we are developing custom Java- and C++-based software tools for the analysis of proteomic and array data.